Anti-GITR antibodies

ABSTRACT

Antibodies to human GITR are provided, as well as uses thereof, e.g., in treatment of proliferative and immune disorders.

This Application is a divisional of U.S. patent application Ser. No.14/261,152, filed Apr. 24, 2014; which is a divisional of U.S. patentapplication Ser. No. 13/388,270, filed Apr. 10, 2012, now U.S. Pat. No.8,709,424; which is the national phase filed under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/US2010/047248, filed Aug. 31,2010; which claims the benefit of U.S. Provisional Patent ApplicationNos. 61/313,955, filed Mar. 15, 2010; 61/307,767, filed Feb. 24, 2010and 61/239,667 filed Sep. 3, 2009; each of which is herein incorporatedby referenced in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to antibodies specific forGlucocorticoid-induced TNF receptor (GITR) and uses thereof. Morespecifically, the invention relates to humanized antibodies thatrecognize human GITR and modulate its activity, particularly in immuneand proliferative disorders.

BACKGROUND OF THE INVENTION

Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNFRsuperfamily, is expressed in many components of the innate and adaptiveimmune system (see, e.g., Hanabuchi et al. (2006) Blood 107:3617-3623;and Nocentini and Riccardi (2005) Eur. J. Immunol. 2005. 35:1016-1022).Its membrane expression is increased following T cell activation(Hanabuchi, supra; and Nocentini and Riccardi, supra); its triggeringco-activates effector T lymphocytes and modulates regulatory T cell(Treg) activity (see, e.g., McHugh, et al. (2002) Immunity 2002.16:311-323; Shimizu, et al. (2002) Nat. Immunol. 3:135-142; Ronchetti,et al (2004) Eur. J. Immunol. 34:613-622; and Tone, et al. (2003) Proc.Natl. Acad. Sci. USA 100:15059-15064.

GITR is activated by GITR ligand (GITRL), which is mainly expressed onAPC and has been suggested to deliver signals by its cytoplasmic domain,although further studies are necessary to define the biochemicalsignaling (Nocentini, supra; Ronchetti, supra; Suvas, et al. (2005) J.Virol. 79:11935-11942; and Shin, et al. (2002) Cytokine 19:187-192).

GITR activation increases resistance to tumors and viral infections, isinvolved in autoimmune/inflammatory processes and regulates leukocyteextravasation (Nocentini supra; Cuzzocrea, et al. (2004) J. Leukoc.Biol. 76:933-940; Shevach et al. (2006) Nat. Rev. Immunol. 6:613-618;Cuzzocrea, et al. (2006) J. Immunol. 177:631-641; and Cuzzocrea et al.(2007) FASEB J. 21:117-129).

The need exists for improved methods and compositions for the treatmentof immune and proliferative disorders, e.g., tumors and cancers, by useof agents that modulate GITR activity. Preferably, such agonists wouldhave a high affinity for the target molecule, and would be able tostimulate GITR signaling at relatively low doses. Preferably, suchmethods and compositions would be highly specific for GITR, and notinterfere with the activity of other receptors. Preferably, such methodsand compositions would employ agonists suitable for modification for thedelivery of cytotoxic payloads to target cells, but also suitable fornon-cytotoxic uses. Preferably, such methods and compositions wouldemploy antibodies modified to limit their antigenicity when administeredto a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the synergistic effect of combined treatment with DTA-1(specific for mGITR; see, e.g., Shimizu, et al. (2002) Nature Immunol.3:135-142) and local irradiation. “CR” means complete regression.

FIG. 2 shows the modules of GITR as determined by the method describedin Naismith and Sprang (1998) Trends Biochem. Sci. 23:74-79. Boldresidues indicate the conformational DTA-1-like epitope as determinedbelow.

SUMMARY OF THE INVENTION

The present invention meets these needs in the art and more by providingagonists of GITR, e.g. humanized anti-GITR antibodies.

In one aspect the invention provides binding compounds, such as anantibodies or fragment thereof, including humanized or chimericrecombinant antibodies, that binds human GITR, comprising an antibodylight chain variable domain, or antigen binding fragment thereof, havingat least one or more CDRs selected from the group consisting of SEQ IDNOs: 56-88 and a heavy chain variable domain, having at least one ormore CDRs selected from the group consisting of SEQ ID NOs: 23-55.

In other embodiments the binding compound of the present inventioncomprises a light chain variable domain and a heavy chain variabledomain, or the antigen binding fragments thereof, described in thepreceding two paragraphs.

In some embodiments, the binding compound comprises a framework region,wherein the amino acid sequence of the framework region is all orsubstantially all of a human immunoglobulin amino acid sequence.

In some embodiments the light chain variable domain comprises a sequenceselected from the group consisting of SEQ ID NOs: 12-22 or a variantthereof. In some embodiments the heavy chain variable domain comprises asequence selected from the group consisting of SEQ ID NOs: 1-11. In yeta further embodiment, the binding compound comprises a light chainvariable domain and a heavy chain variable domain, or the antigenbinding fragments thereof, described in this paragraph.

In other embodiments the binding compound of the present inventioncomprises a light chain variable domain, or an antigen binding fragmentthereof, consisting essentially of a sequence selected from the groupconsisting of SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, and/or a heavy chain variable domain, or an antigen bindingfragment thereof, consisting essentially of a sequence selected from thegroup consisting of SEQ ID NOs: 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110.

In one embodiment, the invention relates to antibodies that are able toblock the binding of a binding compound of the present invention tohuman GITR in a cross-blocking assay. In various embodiments theantibody is able to block binding of human GITR to an antibodycomprising the CDR sequences of antibodies 36E5, 3D6, 61G6, 6H6, 61F6,1D8, 17F10, 35D8, 49A1, 9E5, or 31H6 as disclosed herein. In anotherembodiment, the invention relates to binding compounds that are able toblock GITR-mediated activity, such activities including but not limitedto, co-stimulation of naïve CD4+ T cell proliferation assay.

In some embodiments, the binding compound of the present inventionfurther comprises a heavy chain constant region, wherein the heavy chainconstant region comprises a γ1, γ2, γ3, or γ4 human heavy chain constantregion or a variant thereof. In various embodiments the light chainconstant region comprises a lambda or a kappa human light chain constantregion.

In various embodiments the binding compounds of the present inventionare polyclonal, monoclonal, chimeric, humanized or fully humanantibodies or fragments thereof. The present invention also contemplatesthat the antigen binding fragment is an antibody fragment selected fromthe group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, and adiabody.

The present invention encompasses a method of enhancing an immuneresponse in a human subject comprising administering to a subject inneed thereof an antibody (or a antigen binding fragment thereof)specific for GITR in an amount effective to stimulate GITR signaling. Insome embodiments, the antibody specific for GITR is the humanized orchimeric antibody. In further embodiments, the immune response is ananti-infective or anti-viral response. In certain embodiments, the GITRantibody or antigen binding fragment thereof is co-administered with aTGFβ antibody or local radiation.

The present invention encompasses an isolated nucleic acid encoding thepolypeptide sequence of an antibody embodiment of the binding compoundof the present invention. The nucleic acid can be in an expressionvector operably linked to control sequences recognized by a host celltransfected with the vector. Also encompassed is a host cell comprisingthe vector, and a method of producing a polypeptide comprising culturingthe host cell under conditions wherein the nucleic acid sequence isexpressed, thereby producing the polypeptide, and recovering thepolypeptide from the host cell or medium.

The present invention provides an antibody or antigen binding fragmentthereof, produced by a hybridoma deposited at the American Type CultureCollection (ATCC), wherein the hybridoma is selected from the groupconsisting of PTA-9889, PTA-9890, PTA-9891, PTA-9892, PTA-9893,PTA-10286, PTA-10287, PTA-10288, PTA-10289, PTA-10290, and PTA-10291.

The present invention encompasses an antibody or antigen bindingfragment that binds to human GITR protein, wherein the antibody orantigen binging fragment recognizes an epitope spanning module 3 andmodule 4 of human GITR protein (SEQ ID NO: 89). In certain embodiments,the epitope comprises Gly⁵⁷, Arg⁶⁵, His⁶⁷, Lys⁸⁰, Phe⁸¹, Ser⁸², andGln⁸⁶. In yet other embodiments the antibody cross-blocks at least oneof the antibodies or antibody fragments produced by the hybridomasselected from group consisting of PTA-9889, PTA-9890, PTA-9891,PTA-9892, PTA-9893, PTA-10286, PTA-10287, PTA-10288, PTA-10289,PTA-10290, and PTA-10291.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. Table 15 belowprovides a listing of sequence identifiers used in this application. Allreferences cited herein are incorporated by reference to the same extentas if each individual publication, database entry (e.g. Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.Citation of the references herein is not intended as an admission thatany of the foregoing is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

I. Definitions

The terms “GITR”, “Glucocorticoid-induced TNFR-related protein”,“Activation-inducible TNFR family receptor”, “AITR”, “Tumor necrosisfactor receptor superfamily member 18”, and “TNFSF18” are well known inthe art. The human and mouse GITR nucleotide and polypeptide sequencesare disclosed in WO 98/06842. GenBank® deposits of the human GITR aminosequence (Q9Y5U5) and mouse GITR nucleic and amino acid sequences(AF109216) are also available.

“Proliferative activity” encompasses an activity that promotes, that isnecessary for, or that is specifically associated with, e.g., normalcell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell.“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an agent with animal subject, a cell, tissue,physiological compartment, or physiological fluid. “Treatment of a cell”also encompasses situations where the agent contacts GITR, e.g., in thefluid phase or colloidal phase, but also situations where the agonist orantagonist does not contact the cell or the receptor.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, fully human antibodies, etc. so long as they exhibit thedesired biological activity.

As used herein, the terms “GITR binding fragment,” “binding fragmentthereof” or “antigen binding fragment thereof” encompass a fragment or aderivative of an antibody that still substantially retains itsbiological activity of inducing GITR signaling referred to herein as“GITR inducing activity.” The term “antibody fragment” or GITR bindingfragment refers to a portion of a full length antibody, generally theantigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules, e.g., sc-Fv; andmultispecific antibodies formed from antibody fragments. Typically, abinding fragment or derivative retains at least 10% of its GITR agonistactivity. Preferably, a binding fragment or derivative retains at least25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its GITRagonist activity, although any binding fragment with sufficient affinityto exert the desired biological effect will be useful. It is alsointended that a GITR binding fragment can include variants havingconservative amino acid substitutions that do not substantially alterits biologic activity.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature 352: 624-628 and Marks et al. (1991) J Mol. Biol. 222: 581-597,for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. U.S. Pat. No. 4,816,567;Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079). In one embodiment, the presentinvention provides single domain antibodies comprising two V_(H) domainswith modifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The prefix “hum”,“hu” or “h” is added to antibody clone designations when necessary todistinguish humanized antibodies from parental rodent antibodies. Thehumanized forms of rodent antibodies will generally comprise the sameCDR sequences of the parental rodent antibodies, although certain aminoacid substitutions may be included to increase affinity, increasestability of the humanized antibody, or for other reasons.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The antibodies of the present invention also include antibodies withintact Fc regions that provide full effector functions, e.g. antibodiesof isotype IgG1, which induce complement-dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC) in a targeted cell.

The antibodies of the present invention also include antibodiesconjugated to cytotoxic payloads, such as cytotoxic agents orradionuclides. Such antibody conjugates may be used in immunotherapy inconjunction with anti-GITR treatment, to selectively target and killcells expressing certain antigens on their surface. Exemplary cytotoxicagents include ricin, vinca alkaloid, methotrexate, Psuedomonasexotoxin, saporin, diphtheria toxin, cisplatin, doxorubicin, abrintoxin, gelonin and pokeweed antiviral protein. Exemplary radionuclidesfor use in immunotherapy with the antibodies of the present inventioninclude ¹²⁵I ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹¹At, ¹⁷⁷U, ¹⁴³Pr and ²¹³Bi. See, e.g.,U.S. Patent Application Publication No. 2006/0014225.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” or “rat antibody” refer to an antibody that comprises onlymouse or rat immunoglobulin sequences, respectively. A fully humanantibody may be generated in a human being, in a transgenic animalhaving human immunoglobulin germline sequences, by phage display orother molecular biological methods.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain (Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987)J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or“FR” residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues. Theresidue numbering above relates to the Kabat numbering system and doesnot necessarily correspond in detail to the sequence numbering in theaccompanying Sequence Listing.

“Binding compound” refers to a molecule, small molecule, macromolecule,polypeptide, antibody or fragment or analogue thereof, or solublereceptor, capable of binding to a target. “Binding compound” also mayrefer to a complex of molecules, e.g., a non-covalent complex, to anionized molecule, and to a covalently or non-covalently modifiedmolecule, e.g., modified by phosphorylation, acylation, cross-linking,cyclization, or limited cleavage, that is capable of binding to atarget. When used with reference to antibodies, the term “bindingcompound” refers to both antibodies and antigen binding fragmentsthereof. “Binding” refers to an association of the binding compositionwith a target where the association results in reduction in the normalBrownian motion of the binding composition, in cases where the bindingcomposition can be dissolved or suspended in solution. “Bindingcomposition” refers to a molecule, e.g. a binding compound, incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive, capable of binding to a target.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids are known to those of skill in this artand may often be made even in essential regions of the polypeptidewithout altering the biological activity of the resulting molecule. Suchexemplary substitutions are preferably made in accordance with those setforth in Table 1 as follows:

TABLE 1 Exemplary Conservative Amino Acid Substitutions OriginalConservative residue substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn(N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp;Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys(K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser(S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide may notsubstantially alter biological activity. See, e.g., Watson et al. (1987)Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224(4th Edition).

The phrase “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, that do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a non-limiting example, a bindingcompound that consists essentially of a recited amino acid sequence mayalso include one or more amino acids, including substitutions of one ormore amino acid residues, that do not materially affect the propertiesof the binding compound.

“Effective amount” encompasses an amount sufficient to ameliorate orprevent a symptom or sign of the medical condition. Effective amountalso means an amount sufficient to allow or facilitate diagnosis. Aneffective amount for a particular patient or veterinary subject may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method route and dose of administration andthe severity of side affects. See, e.g., U.S. Pat. No. 5,888,530. Aneffective amount can be the maximal dose or dosing protocol that avoidssignificant side effects or toxic effects. The effect will result in animprovement of a diagnostic measure or parameter by at least 5%, usuallyby at least 10%, more usually at least 20%, most usually at least 30%,preferably at least 40%, more preferably at least 50%, most preferablyat least 60%, ideally at least 70%, more ideally at least 80%, and mostideally at least 90%, where 100% is defined as the diagnostic parametershown by a normal subject. See, e.g., Maynard et al. (1996) A Handbookof SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.;Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ.,London, UK.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resisteradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

The term immune disorder means a disease in which a component of theimmune system of a mammal causes, mediates or otherwise contributes to amorbidity in the mammal. Also included are diseases in which stimulationor intervention of the immune response has an ameliorative effect onprogression of the disease. Included within this term are autoimmunediseases, immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, and immunodeficiencydiseases. Examples of immune-related and inflammatory diseases, some ofwhich are immune or T cell mediated, which can be treated according tothe invention include systemic lupus erythematosis, rheumatoidarthritis, juvenile chronic arthritis, spondyloarthropathies, systemicsclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjogren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory and fibrotic lung diseases such as inflammatory boweldisease (ulcerative colitis: Crohn's disease), gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis, allergic diseases such as asthma,allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includeAIDS (HIV infection), hepatitis A, B, C, D, and E, bacterial infections,fungal infections, protozoal infections and parasitic infections.

The terms “cancer”, “tumor”, “cancerous”, and “malignant” refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma including adenocarcinoma, lymphoma,blastoma, melanoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer such as hepatic carcinomaand hepatoma, bladder cancer, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, myeloma (such as multiple myeloma),salivary gland carcinoma, kidney cancer such as renal cell carcinoma andWilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulvalcancer, thyroid cancer, testicular cancer, esophageal cancer, andvarious types of head and neck cancer.

As cancerous cells grow and multiply, they form a mass of canceroustissue, that is a tumor, which invades and destroys normal adjacenttissues. Malignant tumors are cancer. Malignant tumors usually can beremoved, but they may grow back. Cells from malignant tumors can invadeand damage nearby tissues and organs. Also, cancer cells can break awayfrom a malignant tumor and enter the bloodstream or lymphatic system,which is the way cancer cells spread from the primary tumor (i.e., theoriginal cancer) to form new tumors in other organs. The spread ofcancer in the body is called metastasis (What You Need to Know AboutCancer—an Overview, NIH Publication No. 00-1566; posted Sep. 26, 2000,updated Sep. 16, 2002 (2002)).

As used herein, the term “solid tumor” refers to an abnormal growth ormass of tissue that usually does not contain cysts or liquid areas.Solid tumors may be benign (not cancerous) or malignant (cancerous).Different types of solid tumors are named for the type of cells thatform them. Examples of solid tumors are sarcomas, carcinomas, andlymphomas. Leukemias (cancers of the blood) generally do not form solidtumors (National Cancer Institute, Dictionary of Cancer Terms).

As used herein, the term “primary cancer” refers to the original tumoror the first tumor. Cancer may begin in any organ or tissue of the body.It is usually named for the part of the body or the type of cell inwhich it originates (Metastatic Cancer: Questions and Answers, CancerFacts 6.20, National Cancer Institute, reviewed Sep. 1, 2004 (2004)).

As used herein, the term “carcinoma in situ” refers to cancerous cellsthat are still contained within the tissue where they started to grow,and have not yet become invasive or spread to other parts of the body.

As used herein, the term “carcinomas” refers to cancers of epithelialcells, which are cells that cover the surface of the body, producehormones, and make up glands. Examples of carcinomas are cancers of theskin, lung, colon, stomach, breast, prostate and thyroid gland.

As used herein, the term “isolated nucleic acid molecule” refers to anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences involved inthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences, including rodent (e.g. mouse)and human germline sequences. Any suitable source of unrearrangedimmunoglobulin DNA may be used. Human germline sequences may beobtained, for example, from JOINSOLVER® germline databases on thewebsite for the National Institute of Arthritis and Musculoskeletal andSkin Diseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

To examine the extent of enhancement of GITR activity, for example,samples or assays comprising a given, e.g., protein, gene, cell, ororganism, are treated with a potential activating or inhibiting agentand are compared to control samples without the agent. Control samples,i.e., not treated with agent, are assigned a relative activity value of100%. Inhibition is achieved when the activity value relative to thecontrol is about 90% or less, typically 85% or less, more typically 80%or less, most typically 75% or less, generally 70% or less, moregenerally 65% or less, most generally 60% or less, typically 55% orless, usually 50% or less, more usually 45% or less, most usually 40% orless, preferably 35% or less, more preferably 30% or less, still morepreferably 25% or less, and most preferably less than 20%. Activation isachieved when the activity value relative to the control is about 110%,generally at least 120%, more generally at least 140%, more generally atleast 160%, often at least 180%, more often at least 2-fold, most oftenat least 2.5-fold, usually at least 5-fold, more usually at least10-fold, preferably at least 20-fold, more preferably at least 40-fold,and most preferably over 40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003)Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med. Clin.North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev. GenomicsHum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Small molecule” is defined as a molecule with a molecular weight thatis less than 10 kDa, typically less than 2 kDa, and preferably less than1 kDa. Small molecules include, but are not limited to, inorganicmolecules, organic molecules, organic molecules containing an inorganiccomponent, molecules comprising a radioactive atom, synthetic molecules,peptide mimetics, and antibody mimetics. As a therapeutic, a smallmolecule may be more permeable to cells, less susceptible todegradation, and less apt to elicit an immune response than largemolecules. Small molecules, such as peptide mimetics of antibodies andcytokines, as well as small molecule toxins are described. See, e.g.,Casset et al. (2003) Biochem. Biophys. Res. Commun. 307:198-205;Muyldermans (2001) J. Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol.18:1251-1256; Apostolopoulos et al. (2002) Curr. Med. Chem. 9:411-420;Monfardini et al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues et al.(1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003) Biochem. J.371:603-608; U.S. Pat. No. 6,326,482.

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction that is determinative of the presence of the protein ina heterogeneous population of proteins and other biologics. Thus, underdesignated conditions, a specified ligand binds to a particular receptorand does not bind in a significant amount to other proteins present inthe sample. As used herein, an antibody is said to bind specifically toa polypeptide comprising a given sequence (in this case GITR) if itbinds to polypeptides comprising the sequence of GITR but does not bindto proteins lacking the sequence of GITR. For example, an antibody thatspecifically binds to a polypeptide comprising GITR may bind to aFLAG®-tagged form of GITR but will not bind to other FLAG®-taggedproteins.

The antibody, or binding composition derived from the antigen-bindingsite of an antibody, of the contemplated method binds to its antigenwith an affinity that is at least two fold greater, preferably at leastten times greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with unrelatedantigens. In a preferred embodiment the antibody will have an affinitythat is greater than about 10⁹ liters/mol, as determined, e.g., byScatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239.

“Chronic viral infection” or “persistent viral infection” as usedherein, is meant a viral infection of humans or other animals which isable to infect a host and reproduce within the cells of a host over aprolonged period of time—usually weeks, months or years, without provingfatal. Amongst viruses giving rise to chronic infections and which maybe treated in accordance with the present invention are the humanpapilloma viruses (HPV), Herpes simplex and other herpes viruses, theviruses of hepatitis B and C (HBV and HCV) as well as other hepatitisviruses, the measles virus, all of which can produce important clinicaldiseases, and HIV. Prolonged infection may ultimately lead to theinduction of disease which may be, e.g. in the case of hepatitis C virusliver cancer, fatal to the patient. Other chronic viral infections whichmay be treated in accordance with the present invention include EpsteinBarr virus (EBV), as well as other viruses such as those which may beassociated with tumors, or in the case of animals, various veterinaryviral diseases, for example those of domestic pets or farmyard animalsimportant in agriculture.

The term “antiviral activity” refers to an inhibition of viraltransmission to uninfected cells, inhibition of the replication of avirus, prevention of the virus from establishing itself in a host, orameliorating or alleviating the symptoms of the disease caused by viralinfection. These effects can be evidenced by a reduction in viral loador decrease in mortality and/or morbidity, which assays are describedinfra. An antiviral agent or drug, has antiviral activity and is usefulfor treating persistent or chronic viral infections alone, or as part ofa multi-drug combination therapy.

II. General

The present invention provides engineered anti-GITR antibodies and usesthereof to treat immune disorders, in particular impaired response toinfectious diseases (including viral infections) and cancer.

GITR, also known as TNFRSF18, is a receptor belonging to the TNR-Rsuperfamily. To date, crystal structures of human or mouse GITR are notavailable, however, a modular architecuture of the molecule, based uponstudies described, e.g., in Naismith and Sprang (1998) Trends Biochem.Sci. 23:74-79, can be established. FIG. 2 illustrates that human GITRcan be divided into 6 modules. From the studies below, certainantibodies having agonist activity may have conformational epitopes thatspan modules 3 and 4.

II. Generation of GITR Specific Antibodies

Any suitable method for generating monoclonal antibodies may be used.For example, a recipient may be immunized with GITR or a fragmentthereof. Any suitable method of immunization can be used. Such methodscan include adjuvants, other immunostimulants, repeated boosterimmunizations, and the use of one or more immunization routes. Anysuitable source of GITR can be used as the immunogen for the generationof the non-human antibody of the compositions and methods disclosedherein. Such forms include, but are not limited whole protein,peptide(s), and epitopes generated through recombinant, synthetic,chemical or enzymatic degradation means known in the art. In preferredembodiments the immunogen comprises the extracellular portion of GITR.

Any form of the antigen can be used to generate the antibody that issufficient to generate a biologically active antibody. Thus, theeliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents known in the art. The eliciting antigen may be anisolated full-length protein, a cell surface protein (e.g., immunizingwith cells transfected with at least a portion of the antigen), or asoluble protein (e.g., immunizing with only the extracellular domainportion of the protein). The antigen may be produced in a geneticallymodified cell. The DNA encoding the antigen may genomic or non-genomic(e.g., cDNA) and encodes at least a portion of the extracellular domain.As used herein, the term “portion” refers to the minimal number of aminoacids or nucleic acids, as appropriate, to constitute an immunogenicepitope of the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

Any suitable method can be used to elicit an antibody with the desiredbiologic properties to enhance GITR signaling. It is desirable toprepare monoclonal antibodies (mAbs) from various mammalian hosts, suchas mice, rats, other rodents, humans, other primates, etc. Descriptionof techniques for preparing such monoclonal antibodies may be found in,e.g., Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding(1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) AcademicPress, New York, N.Y. Thus, monoclonal antibodies may be obtained by avariety of techniques familiar to researchers skilled in the art.Typically, spleen cells from an animal immunized with a desired antigenare immortalized, commonly by fusion with a myeloma cell. See Kohler andMilstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogenes, or retroviruses, or other methods known in the art. See,e.g., Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUECULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences that encode a monoclonal antibody or a antigen bindingfragment thereof by screening a DNA library from human B cellsaccording, e.g., to the general protocol outlined by Huse et al. (1989)Science 246:1275-1281.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al. supra; and Ward etal. (1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance that provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989)Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenic mice,see Mendez et al. (1997) Nature Genetics 15:146-156. See also Abgenixand Medarex technologies.

Alternatively, monoclonal antibodies can be produced by enrichment ofclonal populations of B cells isolated from spleens of animals (e.g.,mice, rats, rabbits, etc.) immunized with human GITR (see, e.g.,WO2008045140, U.S. Pat. No. 5,627,052, and US20030186327).

Antibodies or binding compositions against predetermined fragments ofGITR can be raised by immunization of animals with conjugates of thepolypeptide, fragments, peptides, or epitopes with carrier proteins.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective GITR. These monoclonal antibodies will usually bind with atleast a K_(d) of about 1 μM, more usually at least about 300 nM, 30 nM,10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM or better, usually determinedby ELISA or Biacore. Suitable non-human antibodies may also beidentified using the biologic assays described in Examples 5 and 6,below.

Hybridomas corresponding to clones 36E5, 3D6, 61G6, 6H6 and 61F6 weredeposited at the American Type Culture Collection (“ATCC”) under theBudapest Treaty requirements, as PTA-9890, PTA-9889, PTA-9891, PTA-9892,and PTA-9893, respectively, on Mar. 25, 2009.

Hybridomas corresponding to clones 1D8, 17F10, 35D8, 49A1, 9E5, and 31H6were deposited at the ATCC in accordance with the Budapest Treatyrequirements on Aug. 21, 2009, as PTA-10286, PTA-10287, PTA-10288,PTA-10289, PTA-10290, and PTA-10291.

IV. Humanization of GITR Specific Antibodies

Any suitable non-human antibody can be used as a source for thehypervariable region. Sources for non-human antibodies include, but arenot limited to, murine (e.g. Mus musculus), rat (e.g. Rattusnorvegicus), Lagomorphs (including rabbits), bovine, and primates. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance of the desired biological activity. For furtherdetails, see Jones et al. (1986) Nature 321:522-525; Reichmann et al.(1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol.2:593-596.

Methods for recombinantly engineering antibodies have been described,e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S.Pat. No. 4,816,567), Law et al. (European Patent Application PublicationNo. 438310) and Winter (European Patent Application Publication No.239400).

Amino acid sequence variants of humanized anti-GITR antibody areprepared by introducing appropriate nucleotide changes into thehumanized anti-GITR antibody DNA, or by peptide synthesis. Such variantsinclude, for example, deletions from, and/or insertions into, and/orsubstitutions of, residues within the amino acid sequences shown for thehumanized anti-GITR antibody. Any combination of deletion, insertion,and substitution is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of thehumanized anti-GITR antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-GITR antibody polypeptide that are preferred locationsfor mutagenesis is called “alanine scanning mutagenesis,” as describedby Cunningham and Wells (1989) Science 244: 1081-1085. Here, a residueor group of target residues are identified (e.g., charged residues suchas Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with GITR antigen. The amino acidresidues demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, Ala scanning or random mutagenesis isconducted at the target codon or region and the expressed humanizedanti-GITR antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includehumanized anti-GITR antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of thehumanized anti-GITR antibody molecule include the fusion to the N- orC-terminus of humanized anti-GITR antibody of an enzyme or a polypeptidethat increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the humanized anti-GITRantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable loops, but FR alterations are also contemplated.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Yet another type of amino acid variant is the substitution of residuesto provide for greater chemical stability of the final humanizedantibody. For example, an asparagine (N) residue may be changed toreduce the potential for formation of isoaspartate at any NG sequenceswithin a rodent CDR. A similar problem may occur at a DG sequence.Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartateformation may debilitate or completely abrogate binding of an antibodyto its target antigen. Presta (2005) J. Allergy Clin. Immunol. 116:731at 734. In one embodiment, the asparagine is changed to glutamine (Q).In addition, methionine residues in rodent CDRs may be changed to reducethe possibility that the methionine sulfur would oxidize, which couldreduce antigen binding affinity and also contribute to molecularheterogeneity in the final antibody preparation. Id. In one embodiment,the methionine is changed to alanine (A). Antibodies with suchsubstitutions are subsequently screened to ensure that the substitutionsdo not decrease GITR binding affinity to unacceptable levels.

Nucleic acid molecules encoding amino acid sequence variants ofhumanized GITR specific antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofhumanized anti-GITR antibody.

Ordinarily, amino acid sequence variants of the humanized anti-GITRantibody will have an amino acid sequence having at least 75% amino acidsequence identity with the original humanized antibody amino acidsequences of either the heavy or the light chain more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,and most preferably at least 95%, 98% or 99%. Identity or homology withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with thehumanized anti-GITR residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, theantibody is an IgG antibody. Any isotype of IgG can be used, includingIgG₁, IgG₂, IgG₃, and IgG₄. Variants of the IgG isotypes are alsocontemplated. The humanized antibody may comprise sequences from morethan one class or isotype. Optimization of the necessary constant domainsequences to generate the desired biologic activity is readily achievedby screening the antibodies in the biological assays described in theExamples.

Likewise, either class of light chain can be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

Any suitable portion of the CDR sequences from the non-human antibodycan be used. The CDR sequences can be mutagenized by substitution,insertion or deletion of at least one residue such that the CDR sequenceis distinct from the human and non-human antibody sequence employed. Itis contemplated that such mutations would be minimal. Typically, atleast 75% of the humanized antibody residues will correspond to those ofthe non-human CDR residues, more often 90%, and most preferably greaterthan 95%.

Any suitable portion of the FR sequences from the human antibody can beused. The FR sequences can be mutagenized by substitution, insertion ordeletion of at least one residue such that the FR sequence is distinctfrom the human and non-human antibody sequence employed. It iscontemplated that such mutations would be minimal. Typically, at least75% of the humanized antibody residues will correspond to those of thehuman FR residues, more often 90%, and most preferably greater than 95%,98% or 99%.

CDR and FR residues are determined according to the standard sequencedefinition of Kabat. Kabat et al. (1987) Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda Md. SEQID NOs: 1-11 show the heavy chain variable domain sequences of variousrodent anti-human GITR antibodies, and SEQ ID NOs: 12-22 depict thelight chain variable domain sequences.

TABLE 2 Heavy Chain Sequences and Domains ANTIBODY SEQ ID V_(H) HEAVYCHAIN CDR RESIDUES CLONE NO: RESIDUES CDR-H1 CDR-H2 CDR-H3 36H5 1 1-11826-35 50-65 98-107 3D6 2 1-123 26-35 50-66 99-112 61G6 3 1-118 26-3651-66 99-107 6H6 4 1-118 26-35 50-66 99-107 61F6 5 1-119 26-35 50-6699-108 1D8 6 1-122 26-37 52-67 100-111  17F10 7 1-117 26-35 50-65 98-10635D8 8 1-120 26-35 50-65 98-109 49A1 9 1-120 26-35 50-65 98-109 9E5 101-121 26-37 52-67 100-110  31H6 11 1-121 26-37 52-67 100-110 

TABLE 3 Light Chain sequences and Domains V_(L) ANTIBODY SEQ ID RESI-LIGHT CHAIN CDR RESIDUES CLONE NO: DUES CDR-L1 CDR-L2 CDR-L3 36H5 121-113 24-39 54-60  93-101 3D6 13 1-113 24-39 55-61  94-102 61G6 14 1-10824-33 49-55 88-96 6H6 15 1-110 24-35 51-57 90-98 61F6 16 1-113 24-3854-60  93-101 1D8 17 1-118 24-39 55-61  94-102 17F10 18 1-113 24-3450-56 89-97 35D8 19 1-114 24-34 50-56 89-98 49A1 20 1-114 24-34 50-5689-98 9E5 21 1-113 24-34 50-56 89-97 31H6 22 1-113 24-34 50-56 89-97

In one embodiment, CDRs include variants of any single sequence CDRdisclosed herein (SEQ ID NOs: 23-88), in which the variant comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutionsrelative to the disclosed sequence, as determined using the data ofTable 1.

Also contemplated are chimeric antibodies. As noted above, typicalchimeric antibodies comprise a portion of the heavy and/or light chainidentical with, or homologous to, corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity. See U.S. Pat. No. 4,816,567; and Morrisonet al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

Bispecific antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan et al. (1985) Science 229:81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber et al. (1994) J. Immunol.152:5368.

In yet other embodiments, different constant domains may be appended tohumanized V_(L) and V_(H) regions derived from the CDRs provided herein.For example, if a particular intended use of an antibody (or fragment)of the present invention were to call for altered effector functions, aheavy chain constant domain other than IgG1 may be used. Although IgG1antibodies provide for long half-life and for effector functions, suchas complement activation and antibody-dependent cellular cytotoxicity,such activities may not be desirable for all uses of the antibody. Insuch instances an IgG4 or IgG2 constant domain, for example, may beused.

The parental and engineered forms of the antibodies of the presentinvention may also be conjugated to a chemical moiety. The chemicalmoiety may be, inter alia, a polymer, a radionuclide or a cytotoxicfactor. Preferably the chemical moiety is a polymer which increases thehalf-life of the antibody molecule in the body of a subject. Suitablepolymers include, but are not limited to, polyethylene glycol (PEG)(e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol(mPEG). Lee et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEGconjugated single-chain antibodies. Wen et al., (2001) (Bioconj. Chem.12:545-553) disclose conjugating antibodies with PEG which is attachedto a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antibody fragments or the GITR soluble proteins orfragments thereof of the invention may also be conjugated with labelssuch as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F,³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At,²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr and ⁵⁶Fe.

The antibodies and antibody fragments or the GITR soluble proteins orfragments thereof of the invention may also be conjugated withfluorescent or chemilluminescent labels, including fluorophores such asrare earth chelates, fluorescein and its derivatives, rhodamine and itsderivatives, isothiocyanate, phycoerythrin, phycocyanin,allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl,umbelliferone, luciferin, luminal label, isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridimium salt label, anoxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones,biotin/avidin, spin labels and stable free radicals.

Any method known in the art for conjugating the antibody molecules orprotein molecules of the invention to the various moieties may beemployed, including those methods described by Hunter et al., (1962)Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain et al.,(1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. andCytochem. 30:407. Methods for conjugating antibodies and proteins areconventional and very well known in the art.

V. Biological Activity of Humanized Anti-GITR Antibodies

Antibodies having the characteristics identified herein as beingdesirable in a humanized anti-GITR antibody can be screened forinhibitory biologic activity in vitro or suitable binding affinity.Agonist antibodies may be distinguished from antagonist antibodies usingthe biological assay provided at Example 5. Antibodies that exhibitagonist activity will not block the activity of GITR, but will insteadstimulate the response typically mediated by GITR signaling.

To screen for antibodies that bind to the epitope on human GITR bound byan antibody of interest (e.g., those that block binding of GITR), aroutine cross-blocking assay such as that described in ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Antibodies that bind to the same epitopeare likely to cross-block in such assays, but not all cross-blockingantibodies will necessarily bind at precisely the same epitope sincecross-blocking may result from steric hindrance of antibody binding byantibodies bind at overlapping epitopes, or even nearby non-overlappingepitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells (1989) Science 244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human GITR may also be used to determine the functionalepitope for an anti-GITR antibody of the present invention. Mutagenesisstudies, however, may also reveal amino acid residues that are crucialto the overall three-dimensional structure of GITR but that are notdirectly involved in antibody-antigen contacts, and thus other methodsmay be necessary to confirm a functional epitope determined using thismethod.

The epitope bound by a specific antibody may also be determined byassessing binding of the antibody to peptides comprising fragments ofhuman GITR (SEQ ID NO: 41). A series of overlapping peptidesencompassing the sequence of GITR may be synthesized and screened forbinding, e.g. in a direct ELISA, a competitive ELISA (where the peptideis assessed for its ability to prevent binding of an antibody to GITRbound to a well of a microtiter plate), or on a chip. Such peptidescreening methods may not be capable of detecting some discontinuousfunctional epitopes, i.e. functional epitopes that involve amino acidresidues that are not contiguous along the primary sequence of the GITRpolypeptide chain.

The epitope bound by antibodies of the present invention may also bedetermined by structural methods, such as X-ray crystal structuredetermination (e.g., WO2005/044853), molecular modeling and nuclearmagnetic resonance (NMR) spectroscopy, including NMR determination ofthe H-D exchange rates of labile amide hydrogens in GITR when free andwhen bound in a complex with an antibody of interest (Zinn-Justin et al.(1992) Biochemistry 31:11335-11347; Zinn-Justin et al. (1993)Biochemistry 32:6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art. Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York. Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al. eds., Academic Press; U.S. Patent Application PublicationNo. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60;Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.;Roversi et al. (2000) Acta Cryst. D56:1313-1323).

Additional antibodies binding to the same epitope as an antibody of thepresent invention may be obtained, for example, by screening ofantibodies raised against GITR for binding to the epitope, or byimmunization of an animal with a peptide comprising a fragment of humanGITR comprising the epitope sequence. Antibodies that bind to the samefunctional epitope might be expected to exhibit similar biologicalactivities, such as blocking receptor binding, and such activities canbe confirmed by functional assays of the antibodies.

Antibody affinities may be determined using standard analysis. Preferredhumanized antibodies are those that bind human GITR with a K_(d) valueof no more than about 1×10⁻⁷; preferably no more than about 1×10⁻⁸; morepreferably no more than about 1×10⁻⁹; and most preferably no more thanabout 1×10⁻¹° or even 1×10⁻¹¹ M.

The antibodies and fragments thereof useful in the present compositionsand methods are biologically active antibodies and fragments. As usedherein, the term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired the antigenic epitopeand directly or indirectly exerting a biologic effect. As used herein,the term “specific” refers to the selective binding of the antibody tothe target antigen epitope. Antibodies can be tested for specificity ofbinding by comparing binding to GITR to binding to irrelevant antigen orantigen mixture under a given set of conditions. If the antibody bindsto GITR at least 10, and preferably 50 times more than to irrelevantantigen or antigen mixture then it is considered to be specific. Anantibody that “specifically binds” to GITR does not bind to proteinsthat do not comprise the GITR-derived sequences, i.e. “specificity” asused herein relates to GITR specificity, and not any other sequencesthat may be present in the protein in question. For example, as usedherein, an antibody that “specifically binds” to a polypeptidecomprising GITR will typically bind to FLAG®-GITR, which is a fusionprotein comprising GITR and a FLAG® peptide tag, but it does not bind tothe FLAG® peptide tag alone or when it is fused to a protein other thanGITR.

GITR-specific binding compounds of the present invention, such asagonistic GITR specific antibodies, can enhance its biological activityin any manner, including but not limited to increasing the immuneresponse to a microbial infection.

VI. Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions including GITRantibody, the cytokine analogue or mutein, antibody thereto, or nucleicacid thereof, is admixed with a pharmaceutically acceptable carrier orexcipient. See, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions or suspensions. See, e.g., Hardman et al. (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis et al.(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, N Y; Lieberman, et al. (eds.) (1990) PharmaceuticalDosage Forms: Tablets, Marcel Dekker, N Y; Lieberman et al. (eds.)(1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y.

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio of LD₅₀ to ED₅₀. Antibodies exhibiting high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein human. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration.

The mode of administration is not particularly important. Suitableroutes of administration may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof antibody used in the pharmaceutical composition or to practice themethod of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral,intraarterial or intravenous injection.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available. See, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003)New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J.Med. 343:1594-1602.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is substantially derived from the same species as the animaltargeted for treatment (e.g. a humanized antibody for treatment of humansubjects), thereby minimizing any immune response to the reagent.

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, 1-7times per week, one week, two weeks, monthly, bimonthly, etc. Doses maybe provided intravenously, subcutaneously, topically, orally, nasally,rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A preferred dose protocol is one involving the maximal doseor dose frequency that avoids significant undesirable side effects. Atotal weekly dose is generally at least 0.05 μg/kg, 0.2 μg/kg, 0.5μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang et al.(2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J.Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother.52:133-144. The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with autoimmunedisease or pathogen-induced immunopathology and/or a reduction in theseverity of such symptoms that will or are expected to develop. Theterms further include ameliorating existing uncontrolled or unwantedautoimmune-related or pathogen-induced immunopathology symptoms,preventing additional symptoms, and ameliorating or preventing theunderlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with anautoimmune or pathogen-induced immunopathology disease or symptom, orwith the potential to develop such a disease or symptom.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an GITR-specific bindingcompound, e.g. and antibody, that when administered alone or incombination with an additional therapeutic agent to a cell, tissue, orsubject is effective to prevent or ameliorate the autoimmune disease orpathogen-induced immunopathology associated disease or condition or theprogression of the disease. A therapeutically effective dose furtherrefers to that amount of the compound sufficient to result inamelioration of symptoms, e.g., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient administered alone, atherapeutically effective dose refers to that ingredient alone. Whenapplied to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of therapeutic will decrease thesymptoms typically by at least 10%; usually by at least 20%; preferablyat least about 30%; more preferably at least 40%, and most preferably byat least 50%.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, antibody, steroid, chemotherapeutic agent,antibiotic, anti-viral, or radiation, are well known in the art, see,e.g., Hardman et al. (eds.) (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York,N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for AdvancedPractice: A Practical Approach, Lippincott, Williams & Wilkins, Phila.,Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy,Lippincott, Williams & Wilkins, Phila., Pa.

Chemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; XELODA® capecitabine; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON. toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In particular, transforming growth factor (TGF)-β displays an array ofpleiotropic effects in cellular functions such as proliferation,homeostasis, angiogenesis and wound healing. Aberrant regulation ofTGF-β function contributes to cancer progression. Most cancers arecharacterized by excessive transforming growth factor-β production bytumors, which can promote tumor growth and mediateepithelial-to-mesenchymal transition. TGF-β also plays a pivotal rolewithin the immune system maintaining tolerance via the regulation oflymphocyte proliferation, differentiation, and survival. TGF-β has beenproven to be an important suppressive element in enhancing Treg functionand dampening tumor immunity. Administration of TGF-β inhibitors inconjunction with GITR agonists, e.g., antibodies, is contemplated.

Also contemplated is co-administration with anti-viral therapeutics.Anti-virals include any drug that destroys viruses. Antivirals mayinclude interferons which function to inhibits replication of the virus,protease inhibitors, and reverse transcriptase inhibitors or agentscontained in the combination of highly active antiretroviral therapy(HAART) for HIV. Typical veterinary, experimental, or research subjectsinclude monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses,and humans.

VII. Antibody Production

In one embodiment, for recombinant production of the antibodies of thepresent invention, the nucleic acids encoding the two chains areisolated and inserted into one or more replicable vectors for furthercloning (amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.In one embodiment, both the light and heavy chains of a humanizedanti-GITR antibody of the present invention are expressed from the samevector, e.g. a plasmid or an adenoviral vector.

Antibodies of the present invention may be produced by any method knownin the art. In one embodiment, antibodies are expressed in mammalian orinsect cells in culture, such as Chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, babyhamster kidney (BHK) cells, Spodoptera frupperda ovarian (Sf9) cells. Inone embodiment, antibodies secreted from CHO cells are recovered andpurified by standard chromatographic methods, such as protein A, cationexchange, anion exchange, hydrophobic interaction, and hydroxyapatitechromatography. Resulting antibodies are concentrated and stored in 20mM sodium acetate, pH 5.5.

In another embodiment, the antibodies of the present invention areproduced in yeast according to the methods described in WO2005/040395.Briefly, vectors encoding the individual light or heavy chains of anantibody of interest are introduced into different yeast haploid cells,e.g. different mating types of the yeast Pichia pastoris, which yeasthaploid cells are optionally complementary auxotrophs. The transformedhaploid yeast cells can then be mated or fused to give a diploid yeastcell capable of producing both the heavy and the light chains. Thediploid strain is then able to secret the fully assembled andbiologically active antibody. The relative expression levels of the twochains can be optimized, for example, by using vectors with differentcopy number, using transcriptional promoters of different strengths, orinducing expression from inducible promoters driving transcription ofthe genes encoding one or both chains.

In one embodiment, the respective heavy and light chains of a pluralityof different anti-GITR antibodies (the “original” antibodies) areintroduced into yeast haploid cells to create a library of haploid yeaststrains of one mating type expressing a plurality of light chains, and alibrary of haploid yeast strains of a different mating type expressing aplurality of heavy chains. These libraries of haploid strains can bemated (or fused as spheroplasts) to produce a series of diploid yeastcells expressing a combinatorial library of antibodies comprised of thevarious possible permutations of light and heavy chains. Thecombinatorial library of antibodies can then be screened to determinewhether any of the antibodies has properties that are superior (e.g.higher affinity for GITR) to those of the original antibodies. See.E.g., WO2005/040395.

In another embodiment, antibodies of the present invention are humandomain antibodies in which portions of an antibody variable domain arelinked in a polypeptide of molecular weight approximately 13 kDa. See,e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, lowmolecular weight agents provide numerous advantages in terms of ease ofsynthesis, stability, and route of administration.

VIII. Uses

The present invention provides methods for using anti-GITR antibodiesand fragments thereof for the treatment and diagnosis of proliferativeor inflammatory disorders and conditions.

The present invention provides methods for diagnosing the presence of amicrobial infection or cancer by analyzing expression levels of GITR intest cells, tissue or bodily fluids compared with GITR levels in cells,tissues or bodily fluids of preferably the same type from a control. Asdemonstrated herein, an increase in level of GITR expression, forexample, in the patient versus the control is associated with thepresence of cancer.

Typically, for a quantitative diagnostic assay, a positive resultindicating the patient tested has cancer or an infectious disease, isone in which the cells, tissues, or bodily fluids has an GITR expressionlevel at least two times higher, five times higher, ten times higher,fifteen times higher, twenty times higher, twenty-five times higher.

Assay techniques that may be used to determine levels of gene andprotein expression, such as GITR, of the present inventions, in a samplederived from a host are well known to those of skill in the art. Suchassay methods include radioimmunoassays, reverse transcriptase PCR(RT-PCR) assays, quantitative real-time PCR assays, immunohistochemistryassays, in situ hybridization assays, competitive-binding assays,western blot assays, ELISA assays, and flow cytometric assays, forexample, two color FACS analysis for M2 versus M1 phenotyping oftumor-associated macrophages (Mantovani et al., (2002) TRENDS inImmunology 23:549-555).

An ELISA assay initially comprises preparing an antibodies of thepresent invention, specific to GITR, preferably 36E5, 3D6, 61G6, 6H6,61F6, 1D8, 17F10, 35D8, 49A1, 9E5, and 31H6 (collectively “GITRantibodies”). In addition, a reporter antibody generally is preparedthat binds specifically to GITR. The reporter antibody is attached to adetectable reagent such as radioactive, fluorescent or an enzymaticreagent, for example horseradish peroxidase enzyme or alkalinephosphatase.

To carry out the ELISA, at least one of the GITR antibodies describedabove is incubated on a solid support, e.g., a polystyrene dish thatbinds the antibody. Any free protein binding sites on the dish are thencovered by incubating with a non-specific protein, such as bovine serumalbumin. Next, the sample to be analyzed is incubated in the dish,during which time GITR binds to the specific GITR antibody attached tothe polystyrene dish. Unbound sample is washed out with buffer. Areporter antibody specifically directed to GITR and linked tohorseradish peroxidase is placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to GITR. Unattachedreporter antibody is then washed out. Reagents for peroxidase activity,including a calorimetric substrate are then added to the dish.Immobilized peroxidase, linked to GITR antibodies, produces a coloredreaction product. The amount of color developed in a given time periodis proportional to the amount of GITR protein present in the sample.Quantitative results typically are obtained by reference to a standardcurve.

A competition assay may be employed wherein antibodies specific to GITRare attached to a solid support and labeled GITR and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected attached to the solid support can be correlated to a quantityof GITR in the sample.

The above tests may be carried out on samples derived from a variety ofcells, bodily fluids and/or tissue extracts such as homogenates orsolubilized tissue obtained from a patient. Tissue extracts are obtainedroutinely from tissue biopsy and autopsy material. Bodily fluids usefulin the present invention include blood, urine, saliva or any otherbodily secretion or derivative thereof. The term “blood” is meant toinclude whole blood, plasma, serum or any derivative of blood.

Antibodies of the present invention may be used to treat viralinfections. HIV infection is characterized by defects in the generationand maintenance of central memory cells. CD8+ central memory cells havea shorter half-life and are less abundant in HIV-infected individualsthan in controls. Also, the frequency of both CD4+ and CD8+ HIV-specificT cells decreases rapidly after initiation of highly activeantiretroviral therapy (HAART). Co-stimulation on CD4+ by anti-GITR mayprovide a mechanism to increase memory CD8+ response and to contributeto clearance of the virus. It has been shown that treatment ofpersistently Friend virus-infected mice with anti-GITR antibody toameliorate suppression by Tregs significantly improved IFN-γ productionby the CD8+ T cells and allowed a significant reduction in viral loads(Dittmer et al., (2004) Immunity 20: 293-303).

Another characteristic of HIV infection is massive apoptosis of CD4+ Tcells starting early in HIV infection. The progressive apoptoticdeletion of CD4 T cells contributes to weakened HIV-specific cellularimmune responses and to the development of AIDS. GITR co-stimulation hasbeen shown to enhance murine antigen-specific cytokine secretion byprotecting T cells from apoptosis. Lahey et al. (2007) J Infect Dis.196: 43-49) demonstrated that anti-GITR treatment of HIV-specific CD4+ Tcells enhances their cytokine expression and protects them fromapoptosis.

For infections resulting from viral causes, the antibodies of theinvention may be combined by application simulatenous with, prior to orsubsequent to application of standard therapies for treating viralinfections. Such standard therapies vary depending upon type of virus,although in almost all cases, administration of human serum containingantitibodies (e.g., IgA, IgG) specific to the virus can be effective.

Influenza infection results in fever, cough, myalgia, headache andmalaise, which often occur in seasonal epidemics. Influenza is alsoassociated with a number of postinfectious disorders, such asencephalitis, myopericarditis, Goodpasture's syndrome, and Reye'ssyndrome. Influenza infection also suppresses normal pulmonaryantibacterial defenses, such that patient's recovering from influenzahave an increased risk of developing bacterial pneumonia.

Influenza viral surface proteins show marked antigenic variation,resulting from mutation and recombination. Thus, cytolytic T lymphocytesare the host's primary vehicle for the elimination of virus afterinfection. Influenza is classified into three primary types: A, B and C.Influenza A is unique in that it infects both humans and many otheranimals (e.g., pigs, horses, birds and seals) and is the principal causeof pandemic influenza. Also, when a cell is infected by two differentinfluenza A strains, the segmented RNA genomes of two two parental virustypes mix during replication to create a hybrid replicant, resulting innew epidemic strains. Influenza B does not replicate in animals and thushas less genetic variation and influenza C has only a single serotype.

Most conventional therapies are palliatives of the symptoms resultingfrom infection, while the host's immune response actually clears thedisease. However, certain strains (e.g., influenza A) can cause moreserious illness and death. Influenza A may be treated both clinicallyand prophylactically by the administration of the cyclic aminesinhibitors amantadine and rimantadine, which inhibit viral replication.However, the clinical utility of these drugs is limited due to therelatively high incidence of adverse reactions, their narrow anti-viralspectrum (influenza A only), and the propensity of the virus to becomeresistant. The administration of serum IgG antibody to the majorinfluenza surface proteins, hemagglutinin and neuraminidase can preventpulmonary infection, whereas mucosal IgA is required to preventinfection of the upper respiratory tract and trachea. The most effectivecurrent treatment for influenza is vaccination with the administrationof virus inactivated with formalin or β-propiolactone.

After an incubation of 9-11 days, hosts infected with the measles virusdevelope fever, cough, coryza and conjunctivitis. Within 1-2 days, anerythematous, maculopapular rash develop, which quickly spreads over theentire body. Because infection also suppresses cellular immunity, thehost is at greater risk for developing bacterial superinfections,including otitis media, pneumonia and postinfectious encephalomyelitis.Acute infection is associated with significant morbidity and mortality,especially in malnourished adolescents.

Treatment for measles includes the passive administration of pooledhuman IgG, which can prevent infection in non-immune subjects, even ifgiven up to one week after exposure. However, prior immunization withlive, attenuated virus is the most effective treatment and preventsdisease in more than 95% of those immunized. As there is one serotype ofthis virus, a single immunization or infection typically results inprotection for life from subsequent infection.

In a small proportion of infected hosts, measles can develop into SSPE,which is a chronic progressive neurologic disorder resulting from apersistent infection of the central nervous system. SSPE is caused byclonal variants of measles virus with defects that interfere with virionassembly and budding. For these patients, reactivation of T-cells withthe antibodies of the invention so as to facilitate viral clearancewould be desirable.

Hepatitis B virus (HB-V) is the most infectious known bloodbornepathogen. It is a major cause of acute and chronic heptatis and hepaticcarcinoma, as well as life-long, chronic infection. Following infection,the virus replicates in hepatocytes, which also then shed the surfaceantigen HBsAg. The detection of excessive levels of HBsAg in serum isused a standard method for diagnosing a hepatitis B infection. An acuteinfection may resolve or it can develop into a chronic persistentinfection.

Current treatments for chronic HBV include α-inteferon, which increasesthe expression of class I human leukocyte antigen (HLA) on the surfaceof hepatocytes, thereby facilitating their recognition by cytotoxic Tlymphocytes. Additionally, the nucleoside analogs ganciclovir,famciclovir and lamivudine have also shown some efficacy in thetreatment of HBV infection in in clinical trial. Additional treatmentsfor HBV include pegylated α-interferon, adenfovir, entecavir andtelbivudine. While passive immunity can be conferred through parentaladministration of anti-HBsAg serum antibodies, vaccination withinactivated or recombinant HBsAg also confers resistance to infection.The antibodies of the invention may be combined with conventionaltreatments for hepatitis B infections for therapeutic advantage.

Hepatitis C virus (HC-V) infection may lead to a chronic form ofhepatitis, resulting in cirrosis. While symptoms are similar toinfections resulting from Hepatitis B, in distinct contrast to HB-V,infected hosts can be asymptomatic for 10-20 years. Treatment for HC-Vinfection includes the administration of a combination of α-interferonand ribavirin. A promising potential therapy for HC-V infection is theprotease inhibitor telaprevir (VX-960). Additional treatments include:anti-PD-1 antibody (MDX-1106, Medarex), bavituximab (an antibody thatbinds anionic phospholipid phosphatidylserine in a B2-glycoprotein Idependent manner, Peregrine Pharmaceuticals), anti-HPV viral coatprotein E2 antibod(y)(ies) (E.g., ATL 6865-Ab68+Ab65, XTLPharmaceuticals) and Civacir® (polyclonal anti-HCV human immuneglobulin). The antibodies of the invention may be combined with one ormore of these treatments for hepatitis C infections for therapeuticadvantage.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments. The specific embodiments described hereinare offered by way of example only, and the invention is to be limitedby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES Example 1 General Methods

Standard methods in molecular biology are described. Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif. Standard methods also appear in Ausbel et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed. Coligan et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemicalmodification, post-translational modification, production of fusionproteins, glycosylation of proteins are described. See, e.g., Coligan etal. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley andSons, Inc., New York; Ausubel et al. (2001) Current Protocols inMolecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described.Coligan et al. (2001) Current Protcols in Immunology, Vol. 1, John Wileyand Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow andLane, supra. Standard techniques for characterizing ligand/receptorinteractions are available. See, e.g., Coligan et al. (2001) CurrentProtcols in Immunology, Vol. 4, John Wiley, Inc., New York.

Methods for flow cytometry, including fluorescence activated cellsorting detection systems (FACS®), are available. See, e.g., Owens etal. (1994) Flow Cytometry Principles for Clinical Laboratory Practice,John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd)ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry,John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

Standard methods of histology of the immune system are described. See,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available. See, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742;Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren etal. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690.

Example 2 Humanization of Anti-human GITR Antibodies

The humanization of antibodies is described generally, e.g., in PCTpatent application publications WO 2005/047324 and WO 2005/047326.

Briefly, the amino acid sequence of the non-human VH domain (e.g. SEQ IDNOs: 1-11) is compared to a group of five human VH germline amino acidsequences; one representative from subgroups IGHV1 and IGHV4 and threerepresentatives from subgroup IGHV3. The VH subgroups are listed inM.-P. Lefranc (2001) “Nomenclature of the Human Immunoglobulin Heavy(IGH) Genes”, Experimental and Clinical Immunogenetics 18:100-116. Theframework sequences of the human germline sequence with the closestmatch are used to construct a humanized VH domain.

The rodent anti-huGITR antibodies disclosed herein are all of the kappasubclass of VL. The amino acid sequences of the non-human VL domain(e.g. SEQ ID NOs: 12-22) is compared to a group of four human VL kappagermline amino acid sequences. The group of four is comprised of onerepresentative from each of four established human VL subgroups listedin V. Barbie & M.-P. Lefranc (1998) “The Human Immunoglobulin KappaVariable (IGKV) Genes and Joining (IGKJ) Segments”, Experimental andClinical Immunogenetics 15:171-183 and M.-P. Lefranc (2001)“Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”,Experimental and Clinical Immunogenetics 18:161-174. The four subgroupsalso correspond to the four subgroups listed in Kabat et al. (1991-5thEd.) “Sequences of Proteins of Immunological Interest”, U. S. Departmentof Health and Human Services, NIH Pub. 91-3242, pp. 103-130. Theframework sequences of the human germline sequence with the closestmatch are used to construct a humanized VL domain.

Once the target amino acid sequences of the variable heavy and lightchains are determined, plasmids encoding the full-length humanizedantibody may be generated. Plasmid sequences may be altered using Kunkelmutagenesis (see, e.g., Kunkel T A. (1985) Proc. Natl. Acad. Sci. U.S.A82:488-492) to change the DNA sequence to the target humanized antibodysequences. Simultaneously, codon optimization may be performed toprovide for potentially optimal expression.

Antibodies of the present invention can be humanized using a method thatidentifies an acceptor germline sequence for a humanized antibody, andcomprises the steps of: a) identifying a non-human antibody that has thedesired biological activity; b) determining the amino acid sequence of anon-human antibody V_(H) and V_(L) domains; and c) comparing thenonhuman antibody sequence to a group of human germline sequences,wherein the comparison comprises the substeps of: 1) assigning thenon-human V sequences residue numbers according to Kabat supra; 2)delineating the CDR and FR regions in the sequence according to Kabatsupra; 3) assigning a predetermined numerical score at specific residueposition for which the non-human and human antibody germline sequencesare identical; and 4) totaling all of the residue scores to generate atotal score for each human germline sequence; and d) identifying thehuman germline sequence with the highest total residue score as theacceptor germline sequence. In one embodiment, the method furthercomprises the substeps of: 5) assigning a numerical score of 1 for eachFR residue position for which the non-human and human antibody germlinesequences are identical that was not scored in substep (3) to germlinesequences with identical total residue scores after substep (4); 6)totaling all of the residue scores to generate a total score for eachhuman germline sequence. In a specific embodiment, the non-humanantibody is specific for GITR and enhances the biological activity ofGITR. Also provided herein is an antibody generated by the above method.

In one embodiment, the GITR antibody is humanized using the followingmethod. First, the non-human V_(L) and V_(H) domains of the GITRantibody are cloned and sequenced, and the amino acid sequencedetermined. Then, the non-human V_(H) sequence are compared to a groupof three human V_(H) germline amino acid sequences. The three groupscontain one representative from each of subgroups IGHV1, IGHV3 andIGHV4. The V_(H) subgroups are listed in M.-P. Lefranc, Exp. Clin.Immunogenetics, 18:100-116 (2001). Specifically, the comparison with thethree germline sequences begins with the assignment of residue numbersto the non-human V_(H) sequence according to the Kabat numbering system.See Kabat, et al., U. S. Department of Health and Human Services, NIHPub. 91-3242 (5th Ed., 1991). The non-human V_(H) sequence are thenaligned with each of the three human germline sequences. Since the Vgenes only comprise V_(H) residues 1-94, only these residues areconsidered in the alignment. Next, the complementarity-determining (CDR)and framework (FR) regions in the sequence are delineated. CDR and FRare delineated according to the combination of the definitions providedin Kabat, et al., U. S. Department of Health and Human Services, NIHPub. 91-3242 (5th Ed., 1991), and C. Chothia & A. M. Lesk, J. Mol.Biol., 196:901-917 (1987). Therefore, the CDR definition used isresidues 26-35 for CDR1, residues 50-65 for CDR2, and CDR3 is residues95-102 for CDR3 of the V_(H) domain. The next step involves assigning anumerical score at identified residue position where the non-human andhuman sequences are identical. One example of this scoring is shown inTable 4 below.

TABLE 4 Residue # Score Reason 24 3 Affects CDR-H1 27 4 Affects CDR-H1,3* 29 4 Affects CDR-H1* 34 4 Affects CDR-H1* 35 2 VH/VL interface 37 2VH/VL interface 48 3 Affects CDR-H2 49 3 Affects CDR-H2 50 2 VH/VLinterface 58 2 VH/VL interface 60 2 VH/VL interface 63 3 Affects CDR-H267 3 Affects CDR-H2 69 3 Affects CDR-H2 71 4 Affects CDR-H2* 73 3Affects CDR-H1 76 3 Affects CDR-H1 78 3 Affects CDR-H1 94 4 AffectsCDR-H3* max 57 *Noted as affecting CDR conformation in C. Chothia etal., Nature 342: 877-883, (1989).

After the residue positions are assigned a numerical score, all of theresidue scores are totaled. The acceptor germline sequence is the onewith the highest total score. In a case where two or more germlinesequences have identical scores, then add 1 to the total for eachposition where the non-human and human sequences are IDENTICAL for thefollowing FR residues: 1-23, 25, 36, 38-47, 66, 68, 70, 72, 74, 75, 77,and 79-93 (max 60). The residue scores are totaled again, and theacceptor germline sequence is the one with the highest total score. Iftwo or more germline sequences still have identical scores, either onecan be used as the acceptor germline sequence.

If the V_(L) sequence is a member of the kappa subclass of V_(L), thenon-human V_(L) sequence from the GITR specific antibody is compared toa group of four human V_(L) kappa germline amino acid sequences. Thefour sequences are comprised of one representative from each of fourestablished human V_(L) subgroups listed in V. Barbie & M.-P. Lefranc,Exp. Clin. Immunogenetics 15:171-183 (1998) and M.-P. Lefranc, Exp.Clin. Immunogenetics 18:161-174 (2001). The four sequences alsocorrespond to the four subgroups listed in Kabat et al., U. S.Department of Health and Human Services, NIH Pub. 91-3242, pp. 103-130(5th Ed., 1991). The comparison of the non-human sequence to the fourgermline sequences begins with the assignment of residue numbers to thenon-human V_(L) sequence residues according to Kabat et al., U. S.Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed.,1991). The non-human V_(L) sequences are then aligned with each of thefour human germline sequences. Since the V genes only comprise V_(L)residues 1-95, only these residues are considered in the alignment.Next, the complementarity-determining (CDR) and framework (FR) regionsare delineated in the sequence. CDR and FR are delineated according tothe combination of the definitions provided in Kabat et al., U. S.Department of Health and Human Services, NIH Pub. 91-3242 (5th Ed.1991), and C. Chothia & A. M. Lesk, J. Mol. Biol., 196:901-917 (1987).Therefore, the CDR definition used is residues 24-34 for CDR1, residues50-56 for CDR2, and residues 89-97 for CDR3 of the V_(L) domain. Thenext step involves assigning a numerical score at identified residueposition where the non-human and human sequences are identical. Oneexample of this scoring is shown in Table 5 below.

TABLE 5 Residue # Score Reason  2 4 Affects CDR-L1, 3* 25 4 AffectsCDR-L1* 29 4 Affects CDR-L1, 3* 34 2 VL/VH interface 43 2 VL/VHinterface 55 2 VL/VH interface 58 3 Affects CDR-L2 89 2 VL/VH interface91 2 VL/VH interface 94 2 VL/VH interface max 27 *Noted as affecting CDRconformation in C. Chothia et al., Nature 342: 877-883, (1989).

After the residue positions are assigned a numerical score, all of theresidue scores are totaled. The acceptor germline sequence is the onewith the highest total score. In a case where two or more germlinesequences have identical scores, then add 1 to the total for eachposition where the non-human and human sequences are IDENTICAL for thefollowing FR residues: 1-3, 5-23, 35-42, 44-49, 57, 59-88 (max 67). Theresidue scores are totaled again, and the acceptor germline sequence isthe one with the highest total score. If two or more germline sequencesstill have identical scores, either one can be used as the acceptorgermline sequence.

The above parental monoclonal antibodies were humanized using thismethod. SEQ ID NOs: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110are the sequences of variable heavy chains polypeptides, and SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and 111 are the sequencesof the variable light chains.

Example 3 Determining the Equilibrium Dissociation Constant (K_(d)) forAnti-human GITR Antibodies Using KinExA Technology

The equilibrium dissociation constants (K_(d)) for anti human GITRantibodies are determined using the KinExA 3000 instrument. SapidyneInstruments Inc., Boise Id., USA. KinExA uses the principle of theKinetic Exclusion Assay method based on measuring the concentration ofuncomplexed antibody in a mixture of antibody, antigen andantibody-antigen complex. The concentration of free antibody is measuredby exposing the mixture to a solid-phase immobilized antigen for a verybrief period of time. In practice, this is accomplished by flowing thesolution phase antigen-antibody mixture past antigen-coated particlestrapped in a flow cell. Data generated by the instrument are analyzedusing custom software. Equilibrium constants are calculated using amathematical theory based on the following assumptions:

1. The binding follows the reversible binding equation for equilibrium:k _(on)[Ab][Ag]=k _(off)[AbAg]

2. Antibody and antigen bind 1:1 and total antibody equalsantigen-antibody complex plus free antibody.

3. Instrument signal is linearly related to free antibody concentration.

PMMA particles (Sapidyne, Cat No. 440198) are coated with biotinylatedGITR (or a fragment thereof, such as the extracellular domain) accordingto Sapidyne “Protocol for coating PMMA particles with biotinylatedligands having short or nonexistent linker arms.” EZ-link TFP PEO-biotin(Pierce, Cat. No. 21219) is used for biotinylation of GITR, as per themanufacturer's recommendations (Pierce bulletin 0874).

Example 4 Determining the Equilibrium Dissociation Constant (K_(d)) forHumanized Anti-human GITR Antibodies Using BIAcore Technology

BIAcore determinations are performed essentially as described at Example4 of co-pending, commonly assigned U.S. patent application Ser. No.11/511,635 (filed 29 Aug. 2006). Briefly, binding partners areimmobilized on a BIAcore CMS sensor chip using standard amine-couplingprocedure. Kinetic constants for the various interactions are determinedusing BIAevaluation software 3.1. The K_(d) is determined using thecalculated dissociation and association rate constants.

GITR antibodies 36E5, 3D6, 61G6, 6H6, 61F6, 1D8, 17F10, 35D8, 49A1, 9E5,and 31H6 had the following Kd values:

TABLE 6 Affinity measurements of GITR antibodies Analyte Ka (1/Ms) Kd(1/s) Apparent (mAb) Capture antigen (×10⁵) (×10⁻⁶) Kd (pM) 36E5hGITR-hIgG 8.64 177 205 61F6 hGITR-hIgG 11.1 13530 12189 61G6 hGITR-hIgG0.04 69 15602 3D6 hGITR-hIgG 0.95 766 8046 6H6 hGITR-hIgG 6.10 919 15071D8 hGITR-hIgG 4.28 196 458 17F10 hGITR-hIgG 13.1 146 111 35D8hGITR-hIgG 8.01 200 250 49A1 hGITR-hIgG 4078 318 665 9E5 hGITR-hIgG 23.327 12 31H6 hGITR-hIgG 15.7 7 ≤5

Example 5 Bioassays for the Assessment of Activating Anti-GITRAntibodies

The ability of a monoclonal antibody to biologically enhance GITRactivity was assessed by the effect on proliferation of naïve T cells(see, e.g., Ito et al., (2006) PNAS 103(35):13138-43. Naïve CD4⁺ T cellswere isolated from peripheral blood mononuclear cells (PBMC) by Ficollcentrifugation followed by using a naïve CD4 T cell isolation kit fromSTEMCELL Technologies.

In 96-well tissue flat-bottom culture plates, a total of 2×10⁴ freshlypurified naïve CD4 T cells were co-cultured with irradiatedCD32-expressing L cells in the presence of an anti-hGITR antibody or theisotype control, which had been pre-coated with anti-CD3.

Tables 7A and 7B shows the effect of varying doses of anti-GITRantibodies on proliferation of naïve CD4+ T cells (KM4-R63 is an isotypecontrol antibody).

TABLE 7A Ab conc 36E5.A5 JL5.3D6 61G6.B6 6H6.C3 61F6.B9 KM4.R63 (ng/mL)mean SD mean SD mean SD mean SD mean SD mean SD 10000 83784 5854 962059562 135047 7362 91873 4218 93373 4099 29662 7817 2500 71843 7556 10929121713 115725 9792 107347 6049 96296 1233 27007 4572 625 82075 6760111455 5596 75125 5258 120374 10489 105194 5043 25503 4699 156 9713911937 108929 5934 45588 6309 122653 7164 107643 8700 27496 4019 39.190331 6422 124377 26014 40075 2611 103621 2111 107473 2179 25650 197 9.878668 11867 79317 2260 34335 3038 59965 4439 96267 10551 24722 3890 2.447467 9197 43088 1264 28872 2754 35629 3144 61257 3294 24851 1945 0.6131043 6389 33847 1985 32642 3155 30245 12806 37587 1858 29232 7135 0.1526240 8161 30774 7303 35642 4447 33443 8983 31955 2689 26627 1010 0.0427502 4280 33342 3656 30969 1537 38543 8259 34931 5787 31039 3042

TABLE 7B Ab conc 1D8.B5 35D8.B10 49A1.B1 9E5.C1 31H6.B7 17F10.B1 (ng/mL)mean SD mean SD mean SD mean SD mean SD mean SD 1000.0 — — 97278 312778031 5394 83553 6596 87531 1051 89358 12570 416.7 — — 89761 9163 863063807 79685 7730 86915 2652 85702 9724 173.6 90962 3417 96241 3423 935944229 84579 5929 96935 4442 87519 7556 72.3 102810 3353 92371 5048 961265395 85291 16030 98595 7374 83511 5009 30.1 112003 5405 95258 8152 955176187 83407 5503 94683 7610 86986 2717 12.6 115163 6429 86232 5329 860011893 85659 5087 90531 3957 90231 2079 5.2 98161 5423 71405 10471 721921776 77653 5524 79605 7438 77786 5065 2.2 78492 1831 59817 745 61053 23969187 7450 67645 1972 73420 12592 0.9 64788 773 50713 2257 54096 281660922 7139 56972 4470 61950 4598 0.4 60794 5069 52287 5015 54348 101860105 6687 58490 582 58704 7820

Example 5 Treatment of Tumors with TGF-β and GITR Antibodies

Previous studies revealed that gene expression of 4T1 tumors hadincreased levels of TGF-β mRNA. It was hypothesized that immuneco-stimulation by anti-GITR agonist combined with removal of immunesuppression by inhibition of TGF-β signaling would induce synergisticanti-tumor efficacy.

To test this hypothesis, 1.5×10⁵ 4 T1 tumor cells were implantedsubcutaneously at the right flank of Balb/C mice. Four or seven daysafter tumor implantation, a neutralization antibody to murine TGF-β(1D11; Bioexpress) was injected at 100 μgs/200 μL subcutaneously at theneck and repeated every three days for a total of 7 doses. Anti-mouseGITR agonist antibody, DTA-1, was injected at day 7, 14, and 21 at 500μg/200 μL. Tumor volume was measured every three days. As shown in Table8 below, DTA-1 or anti-TGF-β alone has little effect by themselves.Combined treatment induces synergistic effect on either of the startingdays of anti-TGF-β treatment (day 4 or day 7 post tumor implantation).Values are tumor volume (mm³).

TABLE 8 Anti-tumor efficacy by anti-mGITR and/or anti-TGF-β Post tumorIgG2b DTA1 anti-TG-β(D 7) Imp MEAN SD N MEAN SD N MEAN SD N Day 7 57.4764.383 12 55.4614 5.28 12 55.7903 6.85 12 Day 11 160.904 11.87 12 131.56310.67 12 141.827 14.94 12 Day 14 259.459 10.42 12 213.981 20.49 12222.383 18.47 12 Day 18 408.553 24.34 12 340.876 21.30 12 309.058 24.4512 Day 21 627.802 34.23 12 519.613 29.11 12 524.622 44.18 12 Day 25810.945 41.97 12 761.875 45.07 12 699.235 47.79 12 Day 29 1099.43 33.4712 1006.18 39.89 12 908.552 32.36 12 Post tumor DTA1 + anti-TGF-β(D 7)anti-TGF-β(D 4) DTA1 + anti-TGF-β(D 4) Imp MEAN SD N MEAN SD N MEAN SD NDay 7 49.1208 4.24 12 54.551 6.01 10 48.432 6.80 10 Day 11 67.9306 5.9812 140.118 15.54 10 70.202 3.65 10 Day 14 58.5907 5.01 12 193.78 26.5210 39.651 9.522 10 Day 18 100.977 14.92 12 315.661 42.47 10 45.928 20.5910 Day 21 185.615 16.66 12 515.88 55.56 10 92.660 45.67 10 Day 25336.241 27.26 12 718.21 73.21 10 213.447 63.72 10 Day 29 511.284 32.9812 956.36 83.12 10 382.504 94.91 10

Example 6 Antibody-Radiation Combined Treatment of CT26 Tumors

CT26 tumor cells (3×10⁵) were implanted subcutaneously on left flanks ofBalb/c mice. Local irradiation (10 Gy) was applied to tumors that grewto 300 mm³, after observing that DTA-1 alone did not have tumor-killingefficacy. A day after the irradiation, DTA-1 (500 ug) was injectedsubcutaneously on the neck area and repeated every week for a total ofthree doses. Tumor volume was measured every two to five days. In thegroup of 10 mice that underwent local irradiation and DTA-1 combinedtreatment, 5 mice completely rejected the tumors and survived up to 3months. DTA-1 or irradiation alone did not exhibit tumor rejection (see,e.g., FIG. 1).

Example 7 Epitope Mapping of GITR Antibodies

As noted above, DTA-1 is an agonist antibody raised again mouse GITR(see, e.g., Shimizu, et al. supra). DTA-1 has been shown to have potentanti-tumor activities in mouse models of cancer (see, e.g., Cohen, etal. (2006) Cancer Res. 66:4904-4912; Ramirez-Montagut, et al. (2006) J.Immunol. 176:6434-6442; Zhou, et al. (2007) J. Immunol. 179:7365-7375;and Ko, et al. (2005) J. Exp. Med. 202:885-891).

To determine if the antibodies described above bound to a DTA-1-likeepitope on the human GITR protein, the DTA-1 epitope was first mapped onthe mouse GITR protein. Without a crystal structure of human or mouseGITR available, the DTA-1 epitope was determined using standard sitedirected mutagenesis techniques (see, e.g., Kunkel (1985) Proc. Natl.Acad. Sci. 82:488-492) and the general principles of modularity of theTNF-receptor family (see, e.g., Naismith and Sprang, supra).

Once the mouse GITR epitope recognized by DTA-1 was determined, thecorresponding residues on human GITR were changed to the mouse residuesthereby conferring DTA-1 binding to human GITR. From this, it wasdetermined the DTA-1-like epitope on human GITR spanned modules 3 and 4(see FIG. 2), and the human GITR (SEQ ID NO: 89) epitope recognized bytwo of the above-identified antibodies comprised Gly⁵⁷, Arg⁶⁵, His⁶⁷,Lys⁸⁰, Phe⁸¹, Ser⁸², and Gln⁸⁶.

Example 8 Treatment of Viral Infections with Anti-GITR Antibodies

HIV infection is characterized by defects in the generation andmaintenance of central memory cells. CD8+ central memory cells have ashorter half-life and are less abundant in HIV-infected individuals thanin controls. Also, the frequency of both CD4+ and CD8+ HIV-specific Tcells decreases rapidly after initiation of highly active antiretroviraltherapy (HAART). Co-stimulation on CD4+ by anti-GITR may provide amechanism to increase memory CD8+ response and to contribute toclearance of the virus. It has been shown that treatment of persistentlyFriend virus-infected mice with anti-GITR antibody to amelioratesuppression by Tregs significantly improved IFN-γ production by the CD8+T cells and allowed a significant reduction in viral loads (Dittmer etal., (2004) Immunity 20: 293-303).

Another characteristic of HIV infection is massive apoptosis of CD4+ Tcells starting early in HIV infection. The progressive apoptoticdeletion of CD4 T cells contributes to weakened HIV-specific cellularimmune responses and to the development of AIDS. GITR co-stimulation hasbeen shown to enhance murine antigen-specific cytokine secretion byprotecting T cells from apoptosis. Lahey et al. (2007) J Infect Dis.196: 43-49) demonstrated that anti-GITR treatment of HIV-specific CD4+ Tcells enhances their cytokine expression and protects them fromapoptosis.

For infections resulting from viral causes, the antibodies of theinvention may be combined by application simulatenous with, prior to orsubsequent to application of standard therapies for treating viralinfections. Such standard therapies vary depending upon type of virus,although in almost all cases, administration of human serum containingantitibodies (e.g., IgA, IgG) specific to the virus can be effective.

Influenza infection results in fever, cough, myalgia, headache andmalaise, which often occur in seasonal epidemics. Influenza is alsoassociated with a number of postinfectious disorders, such asencephalitis, myopericarditis, Goodpasture's syndrome, and Reye'ssyndrome. Influenza infection also suppresses normal pulmonaryantibacterial defenses, such that patient's recovering from influenzahave an increased risk of developing bacterial pneumonia.

Influenza viral surface proteins show marked antigenic variation,resulting from mutation and recombination. Thus, cytolytic T lymphocytesare the host's primary vehicle for the elimination of virus afterinfection. Influenza is classified into three primary types: A, B and C.Influenza A is unique in that it infects both humans and many otheranimals (e.g., pigs, horses, birds and seals) and is the principal causeof pandemic influenza. Also, when a cell is infected by two differentinfluenza A strains, the segmented RNA genomes of two two parental virustypes mix during replication to create a hybrid replicant, resulting innew epidemic strains. Influenza B does not replicate in animals and thushas less genetic variation and influenza C has only a single serotype.

Most conventional therapies are palliatives of the symptoms resultingfrom infection, while the host's immune response actually clears thedisease. However, certain strains (e.g., influenza A) can cause moreserious illness and death. Influenza A may be treated both clinicallyand prophylactically by the administration of the cyclic aminesinhibitors amantadine and rimantadine, which inhibit viral replication.However, the clinical utility of these drugs is limited due to therelatively high incidence of adverse reactions, their narrow anti-viralspectrum (influenza A only), and the propensity of the virus to becomeresistant. The administration of serum IgG antibody to the majorinfluenza surface proteins, hemagglutinin and neuraminidase can preventpulmonary infection, whereas mucosal IgA is required to preventinfection of the upper respiratory tract and trachea. The most effectivecurrent treatment for influenza is vaccination with the administrationof virus inactivated with formalin or β-propiolactone.

After an incubation of 9-11 days, hosts infected with the measles virusdevelope fever, cough, coryza and conjunctivitis. Within 1-2 days, anerythematous, maculopapular rash develop, which quickly spreads over theentire body. Because infection also suppresses cellular immunity, thehost is at greater risk for developing bacterial superinfections,including otitis media, pneumonia and postinfectious encephalomyelitis.Acute infection is associated with significant morbidity and mortality,especially in malnourished adolescents. Treatment for measles includesthe passive administration of pooled human IgG, which can preventinfection in non-immune subjects, even if given up to one week afterexposure. However, prior immunization with live, attenuated virus is themost effective treatment and prevents disease in more than 95% of thoseimmunized. As there is one serotype of this virus, a single immunizationor infection typically results in protection for life from subsequentinfection.

In a small proportion of infected hosts, measles can develop into SSPE,which is a chronic progressive neurologic disorder resulting from apersistent infection of the central nervous system. SSPE is caused byclonal variants of measles virus with defects that interfere with virionassembly and budding. For these patients, reactivation of T-cells withthe antibodies of the invention so as to facilitate viral clearancewould be desirable.

Hepatitis B virus (HB-V) is the most infectious known bloodbornepathogen. It is a major cause of acute and chronic heptatis and hepaticcarcinoma, as well as life-long, chronic infection. Following infection,the virus replicates in hepatocytes, which also then shed the surfaceantigen HBsAg. The detection of excessive levels of HBsAg in serum isused a standard method for diagnosing a hepatitis B infection. An acuteinfection may resolve or it can develop into a chronic persistentinfection.

Current treatments for chronic HBV include α-inteferon, which increasesthe expression of class I human leukocyte antigen (HLA) on the surfaceof hepatocytes, thereby facilitating their recognition by cytotoxic Tlymphocytes. Additionally, the nucleoside analogs ganciclovir,famciclovir and lamivudine have also shown some efficacy in thetreatment of HBV infection in in clinical trial. Additional treatmentsfor HBV include pegylated α-interferon, adenfovir, entecavir andtelbivudine. While passive immunity can be conferred through parentaladministration of anti-HBsAg serum antibodies, vaccination withinactivated or recombinant HBsAg also confers resistance to infection.The anti-GITR antibodies of the invention may be combined withconventional treatments for hepatitis B infections for therapeuticadvantage.

Hepatitis C virus (HC-V) infection may lead to a chronic form ofhepatitis, resulting in cirrosis. While symptoms are similar toinfections resulting from Hepatitis B, in distinct contrast to HB-V,infected hosts can be asymptomatic for 10-20 years. Treatment for HC-Vinfection includes the administration of a combination of α-interferonand ribavirin. A promising potential therapy for HC-V infection is theprotease inhibitor telaprevir (VX-960). Additional treatments include:anti-PD-1 antibody (MDX-1106, Medarex), bavituximab (an antibody thatbinds anionic phospholipid phosphatidylserine in a B2-glycoprotein Idependent manner, Peregrine Pharmaceuticals), anti-HPV viral coatprotein E2 antibod(y)(ies) (E.g., ATL 6865-Ab68+Ab65, XTLPharmaceuticals) and Civacir® (polyclonal anti-HCV human immuneglobulin). The anti-GITR antibodies of the invention may be combinedwith one or more of these treatments for hepatitis C infections fortherapeutic advantage.

TABLE 9 provides a brief description of the sequences in the sequencelisting. SEQ ID NO.: Description 1 36E5 Heavy Chain Variable 2 3D6 HeavyChain Variable 3 61G6 Heavy Chain Variable 4 6H6 Heavy Chain Variable 561F6 Heavy Chain Variable 6 1D8 Heavy Chain Variable 7 17F10 Heavy ChainVariable 8 35D8 Heavy Chain Variable 9 49A1 Heavy Chain Variable 10 9E5Heavy Chain Variable 11 31H6 Heavy Chain Variable 12 36E5 Light ChainVariable 13 3D6 Light Chain Variable 14 61G6 Light Chain Variable 15 6H6Light Chain Variable 16 61F6 Light Chain Variable 17 1D8 Light ChainVariable 18 17F10 Light Chain Variable 19 35D8 Light Chain Variable 2049A1 Light Chain Variable 21 9E5 Light Chain Variable 22 31H6 LightChain Variable 23 36E5 CDRH1 24 3D6 CDRH1 25 61G6 CDRH1 26 6H6 CDRH1 2761F6 CDRH1 28 1D8 CDRH1 29 17F10 CDRH1 30 35D8 CDRH1 31 49A1 CDRH1 329E5 CDRH1 33 31H6 CDRH1 34 36E5 CDRH2 35 3D6 CDRH2 36 61G6 CDRH2 37 6H6CDRH2 38 61F6 CDRH2 39 1D8 CDRH2 40 17F10 CDRH2 41 35D8 CDRH2 42 49A1CDRH2 43 9E5 CDRH2 44 31H6 CDRH2 45 36E5 CDRH3 46 3D6 CDRH3 47 61G6CDRH3 48 6H6 CDRH3 49 61F6 CDRH3 50 1D8 CDRH3 51 17F10 CDRH3 52 35D8CDRH3 53 49A1 CDRH3 54 9E5 CDRH3 55 31H6 CDRH3 56 36E5 CDRL1 57 3D6CDRL1 58 61G6 CDRL1 59 6H6 CDRL1 60 61F6 CDRL1 61 1D8 CDRL1 62 17F10 CDRL1 63 35D8 CDR L1 64 49A1 CDR L1 65 9E5 CDR L1 66 31H6 CDR L1 67 36E5CDRL2 68 3D6 CDRL2 69 61G6 CDRL2 70 6H6 CDRL2 71 61F6 CDRL2 72 1D8 CDRL273 17F10 CDR L2 74 35D8 CDR L2 75 49A1 CDR L2 76 9E5 CDR L2 77 31H6 CDRL2 78 36E5 CDRL3 79 3D6 CDRL3 80 61G6 CDRL3 81 6H6 CDRL3 82 61F6 CDRL383 1D8 CDRL3 84 17F10 CDR L3 85 35D8 CDR L3 86 49A1 CDR L3 87 9E5 CDR L388 31H6 CDR L3 89 Human GITR 90 Humanized 1D8 VH 91 Humanized 1D8 VL 92Humanized 3D6 VH 93 Humanized 3D6 VL 94 Humanized 6H6 VH 95 Humanized6H6 VL 96 Humanized 9E5 VH 97 Humanized 9E5 VL 98 Humanized 31H6 VH 99Humanized 31H6 VL 100 Humanized 17F10 VH 101 Humanized 17F10 VL 102Humanized 35D8 VH 103 Humanized 35D8 VL 104 Humanized 36E5 VH 105Humanized 36E5 VL 106 Humanized 49A1 VH 107 Humanized 49A1 VL 108Humanized 61F6 VH 109 Humanized 61F6 VL 110 Humanized 61G6 VH 111Humanized 61G6 VL

What is claimed is:
 1. An isolated nucleic acid encoding: a light chainimmunoglobulin variable region that comprises CDR-L1, CDR-L2 and CDR-L3of a light chain immunoglobulin variable region comprising the aminoacid sequence of SEQ ID NO:
 105. 2. The isolated nucleic acid of claim 1encoding: a light chain immunoglobulin variable region that comprisesCDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin variableregion comprising the amino acid sequence of SEQ ID NO: 105, whereinresidue 31 is Q and residue 57 is Q.
 3. The nucleic acid of claim 1encoding: (i) a light chain immunoglobulin variable region thatcomprises CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulinvariable region comprising the amino acid sequence of SEQ ID NO: 105,wherein residue 31 is Q and residue 57 is Q; and (ii) a heavy chainimmunoglobulin variable region that comprises CDR-H1, CDR-H2 and CDR-H3of a heavy chain immunoglobulin variable region comprising the aminoacid sequence of SEQ ID NO:
 104. 4. The nucleic acid of claim 3encoding; (i) a humanized light chain immunoglobulin variable regionthat comprises CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulinvariable region comprising the amino acid sequence of SEQ ID NO: 105,wherein residue 31 is Q and residue 57 is Q; and (ii) a humanized heavychain immunoglobulin variable region that comprises CDR-H1, CDR-H2 andCDR-H3 of a heavy chain immunoglobulin variable region comprising theamino acid sequence of SEQ ID NO:
 104. 5. The nucleic acid of claim 4encoding: (i) a humanized light chain immunoglobulin variable regionthat comprises CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulinvariable region comprising the amino acid sequence of SEQ ID NO: 105wherein residue 31 is Q and residue 57 is Q, and a human light chainconstant region; and (ii) a humanized heavy chain immunoglobulinvariable region that comprises CDR-H1, CDR-H2 and CDR-H3 of a heavychain immunoglobulin variable region comprising the amino acid sequenceof SEQ ID NO: 104, and a human heavy chain constant region.
 6. Thenucleic acid of claim 5 wherein the human light chain constant region iskappa and the human heavy chain constant region is gamma-1.
 7. Thenucleic acid of claim 6 encoding: (i) a humanized light chainimmunoglobulin variable region that comprises CDR-L1, CDR-L2 and CDR-L3of a light chain immunoglobulin variable region comprising the aminoacid sequence of SEQ ID NO: 105 wherein residue 31 is Q and residue 57is Q, and a human kappa light chain constant region; which is operablylinked to a promoter; and (ii) a humanized heavy chain immunoglobulinvariable region that comprises CDR-H1, CDR-H2 and CDR-H3 of a heavychain immunoglobulin variable region comprising the amino acid sequenceof SEQ ID NO: 104, and a human gamma-1 heavy chain constant region;which is operably linked to a promoter.
 8. The nucleic acid of claim 7wherein (i) further comprises a secretory leader and (ii) furthercomprises a secretory leader.
 9. The nucleic acid of claim 8 which isDNA.
 10. An expression vector comprising the nucleic acid of claim 9.11. A host cell comprising the nucleic acid of claim
 9. 12. The hostcell of claim 11 which is a mammalian cell.
 13. The host cell of claim11 which is a Chinese hamster ovary cell.
 14. The host cell of claim 11which is a yeast cell.
 15. The host cell of claim 11 which is a Pichiapastoris cell.
 16. The host cell of claim 11 which is an insect cell.17. The host cell of claim 11 which is a human embryonic kidney 293cell, a mouse myeloma NSO cell, a baby hamster kidney cell, or aSpodoptera frugiperda ovarian cell.
 18. An isolated nucleic acidencoding: a heavy chain immunoglobulin variable region that comprisesCDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin variableregion comprising the amino acid sequence of SEQ ID NO: 104.